An ion-based pacemaker at the pollen tube tip: mechanism and functions in apical growth, guidance and cell-cell communication

花粉管顶端的离子起搏器：顶端生长、引导和细胞间通讯的机制和功能该项目将研究在花粉管中产生的离子和电信号的作用，花粉管是一种高度专业化的细胞，参与开花植物的繁殖。这些细胞代表顶端生长细胞，除了花粉管外，还包括真菌菌丝和哺乳动物神经系统的发育中的神经元。具体地说，这些离子信号是协调受精所需的花粉管生长和功能所必需的。这些结果不仅有助于预测和操纵导致种子生产的细胞行为，具有潜在的农业影响，而且还将发现新的原理，通过这些原理，电信号可以定义细胞的形状、引导和通讯，延伸到植物细胞之外。这项研究将涉及不同STEM领域的发展和整合，包括细胞生物学、遗传学、计算方法和数学建模。根据这项研究开发的数据分析管道和新算法将在R中公开使用，R是一种开放源代码的统计编程语言，可以分析复杂的振荡和细胞生长成像。教育和社会影响包括两名博士后研究人员的专业发展、本科生培训和对当地高中生的指导。此外，一些外展和教育活动是该项目的预期成果，结果和吸引更广泛兴趣的原始方法将向广泛的受众展示。高等植物的受精需要雄配子体(花粉管)生长到雌性组织中，并找到可用的胚珠，最后爆裂交付精子细胞。花粉管是高度极化的细胞，在某些物种中，细胞生长速度是自然界中已知的最快的。这种强劲的增长是由特定的空间和时间编排维持的，通常涉及稳定的振荡，细胞内离子浓度集中在顶端，而细胞外离子通量反过来又产生细胞内离子浓度。本项目要检验的主要假设是存在一个基于离子的中央起搏器，负责花粉管中的微小振荡，(1)由通道活动和膜电位之间的负反馈环产生，(2)在细胞水平上协调宏观过程(如细胞极性、顶端生长、引导)，(3)参与正在生长的花粉管之间的细胞间竞争/合作，以及(4)影响与雌配子体的细胞间通讯。第一个目标是使用定制的电生理学工具和遗传编码探针(钙、氢、氯和膜电位)的实时成像来表征所有参数的振荡。这将使不同物种/生态型的签名与拟南芥中编码通道和转运蛋白的基因家族的单个和多个突变之间的比较成为可能。振荡的特征将通过为该项目开发的数据分析工具来确定，从而可以建立数学模型。然后，具体的预测将从不同程度的微分方程式模型中得出，而实验验证将通过使用突变、药理学和体内受精方案等测试预测来实现。

An ion-based pacemaker at the pollen tube tip: mechanism and functions in apical growth, guidance and cell-cell communicationThe project will study the role of ion and electric signals generated within the pollen tube, a highly specialized cell involved in the reproduction of flowering plants. These cells are representative of apical growing cells, a group that in addition to pollen tubes, includes fungal hyphae and developing neurons of the mammalian nervous system. Specifically these ion signals are needed to coordinate pollen tube growth and function required for fertilization. The results will not only contribute to predicting and manipulating cellular behavior leading to seed production, with potential agricultural impacts, but also to discovering new principles by which electric signals can define cell shape, guidance and communication extending beyond plant cells. This investigation will entail the development and integration of different STEM areas, including cell biology, genetics, computational methods and mathematical modeling. A data analysis pipeline and novel algorithms developed from the research will be made publicly available in R, an open-source statistical programming language, allowing analysis of complex oscillations and cell growth imaging. Educational and societal impacts include professional development of two postdoctoral researchers, undergraduate student training, and mentoring of local high school students. In addition, a number of outreach and educational activities are expected outcomes of the project, where the results and original approaches appealing to broader interests will be presented to a wide variety of audiences.Fertilization in higher plants requires the male gametophyte (pollen tubes) to grow into the female tissues and locate available ovules, finally bursting to deliver the sperm cells. Pollen tubes are highly polarized cells and in some species have the fastest cellular growth rates known in nature. This vigorous growth is sustained by specific spatial and temporal choreographies, often involving stable oscillations, of intracellular ion concentrations focused at the tip, which in turn are generated by extracellular ion fluxes. The main hypothesis to be tested in this project poses the existence of an ion-based central pacemaker responsible for minute-range oscillations in pollen tubes, which (1) are generated by a negative feedback loop between channel activity and membrane potential, (2) coordinate macroscopic processes at the cellular level (e.g. cell polarity, apical growth, guidance), (3) are involved in cell-cell competition/cooperation between growing pollen tubes and (4) impact cell-cell communication with the female gametophyte. The first goal is to characterize oscillations in all parameters using custom-made electrophysiology tools and live imaging with genetically encoded probes (Ca2+, H+, Cl- and membrane potential). These will allow comparison between signatures of different species/ ecotypes and single and multiple mutants of gene families coding for channels and transporters in Arabidopsis. The features of the oscillations will be determined with data-analysis tools developed for the project, allowing the formulation of mathematical models. Specific predictions will then be derived from differential equation models in varying levels of detail, whereas experimental validation will be achieved by testing the predictions with mutations, pharmacology and in vivo fertilization protocols, among others.